This project serves as the ligand/drug development component of a comprehensive collaborative effort to study the endocannabinoid sites of action under the auspices of a Program Reject (PP). High affinity covalent and non-covalent ligands will be utilized to obtain detailed structural information on the active sites of the CB1 and CB2 cannabinoid receptors (CBRs). To enhance our ability to develop ligands with high CB1 or CB2 selectivities, we shall also test our novel ligands for their abilities to interact with key endocannabinoid targets involved in endocannabinoid deactivation including the two hydrolytic enzymes, Fatty Acid Amide Hytlrolase (FAAH) and Monacyl Glycerol Ligase (MGL), the oxidative enzyme Cyclooxygenase-2 (COX-2), and the Anandamide Transport system (ANT). The ligands will be utilized to: (a) probe the CB1 and CB2 binding sites;(b) in vivo image the CB1 receptor in mammalian brains (imaging of FAAH will be a later goal);(c) explore the pharmacophoric requirements within different classes of ligands for CB1 and CB2 desensitization as well as receptor activation, deactivation and dimerization. The new compounds represent structurally diverse classes of cannabinergic agents, including classical and non- classical cannabinoids, aminoalkylindoles, anandamide and 2-arachidonoyl glycerol analogs and pyrazole CB1 and CB2 antagonists. The ligands are designed as photoactivatable or electrophilic, irreversible probes or as high affinity reversible probes. Covalent receptor labeling will be carried out in baculovirus cell culture preparations expressing epitope-tagged CB1 and CB2. Receptor expression, purification and characterization will utilize affinity chromatographic and mass spectroscopic methods. Information on ligand- receptor interactions will be used for the development of non-opioid, non-addictive central and peripheral analgesics, as well as medications designed to combat the ill effects of cannabis and other drugs of abuse. Such medications aim at addressing the drug addiction epidemic and will be of great medical value and social benefit.